Qualitative characterization of antibody responses to single and multiple Brugia pahangi infections in jirds

Antibody responses of jirds, singly and multiply inoculated with Brugia pahangi infective larvae (L3), to soluble somatic extracts of adult parasites were characterized by western blot analysis. Forty-two protein bands ranging in molecular weight from 12 to 160 kDa were recognized by sera from infected jirds. Antibody recognition of individual B. pahangi antigen bands in this assay appears to be independent of antibody enzyme-linked immunosorbent assay (ELISA) titers to crude parasite extract, severity of lymphatic lesions, levels of microfilaremia, numbers of L3 inoculated, or numbers of adult parasites in individual jirds. Antibody recognition of protein bands with molecular weights of 37 kDa, 21 kDa, and 17 kDa, however, did temporally correspond with certain parasitological and pathologic events. Antibody against the 37-kDa protein band first was identified at the onset of patency, reaching a 90% prevalence rate by 90 days postinfection (DPI). The prevalence of this antibody remained high. Antibody recognition of the 21-kDa protein band first occurred at 90 DPI and gradually increased in prevalence during the course of infection temporally similar to the increase in microfilaremia. Recognition of the 17-kDa protein band first occurred at 48 DPI, reached a maximum prevalence of 80% at 90 DPI, and decreased to a minimal prevalence by 160 DPI. Prevalence of antibody responses to the 17-kDa protein band corresponded temporally with the kinetics of the rise and fall of numbers of intralymphatic thrombi. The patterns of antibody response to these 3 bands were similar in both singly and multiply inoculated animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Brugia pahangi: Immunization with early L3 ES alters parasite migration,and reduces microfilaremia and lymphatic lesion formation in gerbils (Meriones unguiculatus)

Previous studies have shown that intradermally (ID) injected Brugia pahangi L3s migrate through various tissues and into the lymphatics of gerbils in a distinct pattern. Excretory/secretory products (ES) produced at the time of invasion of B. pahangi are likely to be important in this early migration phase of the parasite life cycle in their rodent host. Hence, early L3 ES was collected from 24 h in vitro cultures of B. pahangi L3 larvae and used in immunization experiments to investigate the effect of immunity to early L3 ES on worm migration, survival and development of B. pahangi. Immunization of gerbils with ES in RIBI adjuvant produced antibodies to numerous ES proteins eliciting a strong humoral response to ES and indirect fluorescent antibody (IFA) assay using anti-ES serum recognized the ES proteins on the surface of B. pahangi L3 larvae. Following ES immunization, gerbils were challenged either ID or intraperitoneally (IP) with 100 L3s of B. pahangi and euthanized at 3 or 106 days post inoculation (DPI). Immunization with early ES slowed the migration of ID inoculated L3 at 3 DPI and significantly altered the locations of adult worms at 106 DPI. Immunization did not induce protection in any treatment group. However, immunized animals had significantly fewer microfilariae per female worm suggesting the antigens in ES are important in microfilariae development or survival in the host. The number of lymphatic granulomas was also significantly reduced in ES immunized animals. It is important to note that microfilariae serve as a nidus in these granulomas. Our results shows immunization with early Brugia malayi L3 ES alters the worm migration, affects circulating microfilarial numbers and reduces lymphatic granulomas associated with B. pahangi infection in gerbils.     

Brugia pahangi: effects of duration of infection and parasite burden on lymphatic lesion severity, granulomatous hypersensitivity, and immune responses in jirds (Meriones unguiculatus)

The effects of Brugia pahangi infection duration and parasite burden on parasite-associated inflammatory and immune responses were determined over a 181-day period in jirds receiving from one to eight inoculations of infective larvae. Multiple infections did not produce a protective resistance to reinfection as determined by adult worm recovery at necropsy. Intralymphatic granulomatous lesions, lymph thrombi, were first seen at 48 days post initial inoculation (DPI). The numbers of lymph thrombi reached peak levels in singly inoculated jirds at 90 DPI and significantly decreased to low levels by 160 DPI. The ratio of lymph thrombi to adult worms recovered from the spermatic cord lymphatics followed a similar pattern. Sizes of renal lymph nodes, which drain lymphatics containing parasites, followed a temporal pattern of increase and decrease similar to that of lymph thrombi numbers. Peak granuloma areas around antigen-coated beads embolized in lungs were seen at 27 DPI. Granuloma areas around antigen-coated beads began to decrease after 69 DPI and reached sizes not significantly different from uninfected controls by 118 DPI. Multiple inoculations of infective larvae and increasing worm burdens did not affect the pattern of granulomatous response to antigen-coated beads. Eosinophilia of singly and multiply infected jirds peaked at 26 DPI. Eosinophilia of singly infected jirds returned to normal levels by 103 DPI but those of multiply infected jirds remained elevated until 160 DPI. Lymph node cell blastogenic responses to antigen were greater than those of splenocytes at all time intervals measured. However, significant differences in stimulation indexes between groups with different infection durations were not seen with either cell type. Antibody responses to somatic adult worm antigen as measured by ELISA reached near peak levels by 48 DPI and remained elevated for the course of the study in all infected jirds. The decrease in lymphatic lesion severity seen in chronically infected jirds temporally corresponds to the decrease in granulomatous reactivity measured around antigen-coated beads embolized in the lungs. This observation suggests that host and/or parasite factors associated with these two phenomena may be similar. Although these decreases may be the result of down-regulated immune responses, corresponding decreases in antibody levels and blastogenesis of lymphocytes stimulated by crude worm extracts were not observed in chronic infections.     

The anthelmintic activity of a novel organic arsenical, R7/45, upon Brugia pahangi in Meriones unguiculatus

The new organic arsenical R7/45 is a rapidly acting and very potent anthelmintic against adult Brugia pahangi in jirds. Against adult worms implanted into the peritoneal cavity 5 subcutaneous (SC) injections at 2.5 mg/kg of R7/45 killed 100% of adult worms. A single dose SC of 20 mg/kg was 100% effective and 10 mg/kg 76% effective against adult worms. When jirds were autopsied at different times after treatment at 20 mg/kg SC 89% of worms were dead within three days. R7/45 was not active when given by stomach intubation. Pretreatment of jirds with R7/45 had no effect on adult worms subsequently implanted into jirds. R7/45 was highly active against third and fourth stage larvae of B. pahangi in jirds.     

Brugia pahangi: granulomatous lesion development in jirds following single and multiple infections

The development of adult worm burdens and microfilaremias were determined in jirds which received 2, 3, or 4 subcutaneous inoculations of 50 Brugia pahangi infective larvae. Parasite burdens in multiply inoculated jirds were compared to those in four different groups of jirds which received single inoculations of 50 infective larvae. One of each of these singly inoculated groups was infected on the same day that one of the inoculations was given to the multiply infected jirds. Thus, the duration of the infections in the four groups of jirds receiving one inoculation was 54, 118, 189, and 254 days. The development of lymphatic lesions and granulomatous hypersensitivity to B. pahangi antigen was assessed in all jirds at necropsy. The percentage recoveries of adult worms and their locations did not differ in the singly inoculated jirds with infections of different durations. A protective resistance to reinfection, as measured by adult worm recovery in multiply infected jirds, did not occur. The lymphatic lesion scores and numbers of intralymphatic thrombi was greatest in singly inoculated jirds examined 54 days after infection. Pulmonary granuloma areas around adult filarial antigen coated beads embolized in the lungs of jirds 3 days prior to necropsy were also greatest in singly inoculated jirds examined 54 days after infection. Using criteria of lesion scores and lymph thrombi numbers to assess lymphatic lesion severity, a decrease in lesion severity as well as pulmonary granuloma size around antigen coupled beads was seen by 118 days after infection in singly inoculated jirds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies of the effect of contralateral versus ipselateral challenge of rats primed on one side with Strongyloides ratti

The initial pathway of skin penetrating larvae of Strongyloides ratti inside the host is not systemic and could well involve local components of the lymphatic system. The experiments described were an attempt to detect an effect on immunity depending on whether larvae of a challenge infection were committed to a pathway through heavily primed or lightly primed lymph nodes. Female rats were immunised by subcutaneous injection into the right forearm of 10,000 [corrected] heat killed, or 1000 live, third stage larvae of S. ratti. Animals given living parasites were placed on a diet containing 0.1 or 0.2% thiabendazole 48h or 36h after infection. Challenge infections of less than 100 larvae ('exact' doses) were applied on day 21 to either the right or left flank and, similarly, to controls that had received the anthelmintic but not the priming infection. Heat killed parasites elicited no response at all. Priming with live larvae stimulated a significant immunity (16% and 37% depression in 2 experiments), but there was no difference between rats whose challenge was on the same side as the priming dose and those which had the contralateral treatment. The significance of these results to theories of pathfinding in general, and to practical immunology, is discussed.     

Zoonotic Brugia pahangi filariasis in a suburbia of Kuala Lumpur City, Malaysia

Five local Malaysian patients with clinical manifestations consistent with lymphatic filariasis were referred to our medical centre between 2003 and 2006. Although no microfilariae (mf) were detected in their nocturnal blood samples, all were diagnosed to have lymphatic filariasis on the basis of clinical findings and positive serology results. PCR on their blood samples revealed that two of the patients were infected with Brugia pahangi, an animal filarial worm hitherto not known to cause human disease in the natural environment. All the patients were successfully treated with anti-filarial drugs: four patients were treated with a combination of diethylcarbamazine (DEC) and albendazole, and one with doxycycline. Four of them were residents of Petaling Jaya, a residential suburbia located 10 km southwest of Kuala Lumpur city, Malaysia. The fifth patient was a frequent visitor of the suburbia. This suburbia has no history or record of B. malayi infection. The most likely vector of the worm was Armigeres subalbatus as extensive entomological surveys within the suburbia revealed only adult females of this mosquito species were infected with B. pahangi larvae. Wild monkeys caught in the suburbia were free from B. pahangi mf, but domestic cats were mf positive. This suggests that infected cats might be the source of the zoonotic infection in the suburbia.     

Exogenous nucleosides are required for the morphogenesis of the human filarial parasite Brugia malayi

The nematode parasites Wuchereria bancrofti, Brugia malayi, and B. timori cause a human disease known as lymphatic filariasis, which afflicts approximately 120 million people worldwide. The parasites enter the human host from the mosquito as L3 or infective larvae and subsequently differentiate through 2 molts. In this communication, I report that B. malayi and B. pahangi depend on an exogenous source of at least 1 purine and 1 pyrimidine nucleoside to complete the L3 to L4 molt. The requirement for exogenous nucleosides opens the door for possible chemotherapeutic intervention.     

Induction of lymphatic lesions by Brugia pahangi in jirds with large and small preexisting homologous intraperitoneal infections

The effect of intraperitoneal (i.p.) infections induced by inoculations of 30 or 150 Brugia pahangi third-stage larvae (L3) on the development of infections and lymphatic lesions induced by subsequent homologous subcutaneous (s.c.) inoculations were compared in the present study. Lymphatic lesion severity, as judged by the numbers of lymph thrombi present, and lymphatic lesion scores were significantly reduced in both groups of jirds with existing i.p. infections. The numbers of adult worms that developed, locations of these worms, and the subsequent microfilaremias did not differ significantly between groups. All jirds with i.p. infections developed similar antibody titers to crude somatic adult antigen as measured by ELISA. These levels did not change following s.c. infections. Immediate and delayed footpad swelling responses were also similar in all groups. Results of these experiments support and extend previous studies indicating that i.p. infections of B. pahangi induce a hyporesponsive state in jirds to subsequent s.c. infections without significantly affecting the subsequent parasite burden. This effect appears to be independent of the numbers of L3 inoculated i.p. prior to lymphatic-induced infection. Circulating antibody titers and footpad swelling responses to B. pahangi antigen were not reduced in jirds with the hyporesponsive lymphatic inflammatory response and do not correlate with this condition.     

Brugia pahangi: in vivo tissue migration of early L3 alters gene expression

Events occurring during early filarial nematode migrations are central to parasite establishment but rarely studied. Brugia pahangi larvae injected intradermal (ID) into the hind limb of the gerbil (Meriones unguiculatus) can be recovered from the popliteal lymph node (POP) at 3 days post-infection (DPI). They have been designated migrating larvae (IDL3). Alternatively, L3 recovered at 3DPI from the peritoneal cavity (IPL3) do not migrate. Subtracted cDNA libraries using IDL3 and IPL3 revealed distinct gene profiles between IDL3 and IPL3. Troponin-c was significantly upregulated in IDL3, while Cathepsin L was significantly increased in IPL3. Differences in mRNA levels were also observed with these and other genes between IDL3, IPL3 and L3 isolated from mosquitoes (VL3). These data suggest that migratory activity, exposure to potentially different host environments and/or host location may be important external factors in influencing larval gene expression.     

Studies with Brugia pahangi. 15. Cobalt 60 irradiation of the worm

Infective larvae of Brugia pahangi were irradiated at 10, 25 or 45 krads by means of a Cobalt 60 source. In cats, 10 krads caused the worms to be stunted and sterile but allowed them to become 5th stage, migrate posteriorly into the afferent lymphatic, and produce pathology. 25 krads prevented the worms from developing beyond the early fourth stage and from migrating away from the popliteal lymph node. No gross pathological reacions were evident. 45 krads produced the same effects as 25 krads but the longevity of the worms was much reduced.     

Induction of host resistance to Brugia pahangi in jirds (Meriones unguiculatus) protected by chemoprophylaxis

Jirds were given a chemoprophylactic inoculation of flubendazole (FMBZ) and then five injections of infective larvae of Brugia pahangi whilst still protected by the FMBZ. When the drug was thought to be non-effective the jirds (and controls) were given a challenge infection of B. pahangi larvae. By comparison with control jirds the treated-infected-challenged jirds had 40% fewer adult worms. The control treated-challenged jirds contained mostly sterile female worms showing that they were still partially protected by FMBZ but worms numbers were not significantly reduced as compared with untreated controls.     

Extracellular proteases of Onchocerca

Two important events in infection by Onchocerca parasites involve cutaneous tissue migration by larval stages. L3 larvae migrate from the blackfly bite site to subcutaneous locations for adult development, and microfilariae from subcutaneous nodules to distant regions of the skin and sometimes the eye. By analogy to other tissue-invasive helminth larvae, it has been proposed that migration of Onchocerca larvae through cutaneous tissue is facilitated by secretion of proteolytic enzymes. To test this hypothesis, neutral protease activity capable of degrading a model of cutaneous extracellular matrix was assayed using live L3 larvae of O. lienalis and microfilariae of O. cervicalis and O. cervipedis. Five hundred L3 larvae degraded most of the matrix within 24 hr of incubation. Substrate gel electrophoresis and other protease assays showed a 43-kDa serine elastase was secreted by O. lienalis L3 larvae. Larvae and adults of the free-living nematode, Caenorhobditis elegans, by contrast, did not secrete neutral proteases and large numbers of motile C. elegans juveniles and adults produced no degradation of the extracellular matrix. Expression of Onchocerca neutral protease activity was stage specific. No protease activity corresponding to that seen in L3 larvae was found in adult worms. Microfilariae of O. cervicalis and O. cervipedis produced both a serine and a metalloprotease, but the level of protease activity of these microfilariae was substantially lower than that of L3 larvae, and no significant protease activity was detected in extracts of O. lienalis microfilariae. Uterine microfilariae of O. cervicalis had different protease species than skin microfilariae, suggesting that changes in protease expression parallel other morphologic and biochemical changes in the development of skin microfilariae. The serine protease of L3 larvae probably plays an important parasitic function, facilitating L3 migration from the blackfly bite site to distant regions of the body where adults will develop and form nodules. The protease activity of microfilariae, while individually considerably less than that of L3 larvae, may still contribute to the tissue destruction seen with heavy skin densities of microfilariae.     

Brugia pahangi and Wolbachia: the kinetics of bacteria elimination,worm viability,and host responses following tetracycline treatment

Wolbachia spp., first reported from filariae nearly 30 years ago, have been suggested to contribute to the pathogenesis associated with human filarial infection. Tetracycline has been used to cure filariae of Wolbachia, as a novel means of chemotherapeutic treatment for both ocular and lymphatic filariasis. Tetracycline treatment of L4 or adult Brugia pahangi in vivo resulted in Wolbachia clearance. Less tetracycline was required to clear Wolbachia when treatment began at the L4 stage, compared with adults. Female worms died earlier than male worms when tetracycline was administered at the L4 stage. In all cases, Wolbachia clearance was closely associated with worm death. Worm recoveries decreased following the L4-L5 molt, suggesting tetracycline does not interrupt molting in this model system. Despite worm death and the assumed release of both bacterial- and worm-derived molecules, differences in inflammatory cell population and T cell cytokine mRNA profiles were negligible between tetracycline-treated and non-treated B. pahangi infected gerbils. These data suggest the contribution of Wolbachia to the in vivo induction of the gerbil immune response to B. pahangi may be small.     

The effect of method of infection on the pathway of juvenile Strongyloides ratti in the host

Subcutaneous injection of the larvae is the almost universally adopted means of initiating experimental infections of skin-invading roundworms but, so far, the possibility that this procedure introduces artefacts of one kind or another has not been critically studied. Experiments described in this paper were used to compare the effect of (a) injection and (b) skin application, of a small, precisely counted ('exact') dose of larvae. Results with two strains of S. ratti showed that the same proportion of the dose developed to adults in the intestines of rats irrespective of the method. With the same exact dose technique it has been shown that milk-borne infection of the pups of lactating rats is not an artefact produced by injection. Large doses (mean 4000) of larvae of the homogonic strain of S. ratti carrying a radioactive label of 75Se were tracked in their migration to the mammary gland following injection or skin application at two different sites on the right-hand side of nursing mother rats. The broad conclusion of earlier work in this laboratory using injection, that larvae move by a local route and not a systemic one, was supported by the results. The detailed distribution of the label and of unlabelled worms of the heterogonic strain in families was, however, different for the two methods, indicating that subtle variations in pathway can be brought about by the use of injection. If migration involves the lymphatic system, then the interpretation of immunological experiments in terms of lymphatic anatomy must take account of such procedural effects. The extent to which these results contribute to theories of migration in Strongyloides ratti is discussed.     

A novel gene from Brugia sp. that encodes a cytotoxic fatty acid binding protein allergen recognized by canine monoclonal IgE and serum IgE from infected dogs

Brugia pahangi infection of dogs is a well characterized model of human lymphatic filariasis in which sera consistently show IgG or IgE reactivity to a 35-kDa antigen. Using dog lymph node B cells, we previously established a heterohybridoma cell line producing canine monoclonal IgE (cmAb 2.39) that activates and degranulates canine mast cells, and specifically recognizes a 35-kDa B. pahangi antigen. By affinity purification and sequencing of the native protein from B. pahangi adults, a 19-amino acid sequence was obtained; the derived nucleotide sequence showed homology to a Brugia malayi and 2 related Onchocerca volvulus expressed sequence tag (EST) clones from the Filarial Genome Project database. Consensus primers amplified a 244-bp product from adult and infective larval stage cDNA libraries of B. malayi, O. volvulus, and Wuchereria bancrofti, but not from those of nonfilarial nematodes. The B. malayi EST clone only showed nucleotide sequence homology to O. volvulus EST sequences. A 684-bp region from the open reading frame was expressed as a glutathione S-transferase fusion protein designated BmAl-1. CmAb 2.39, as well as serum IgE from dogs infected with B. pahangi and canine filarial heartworm, Dirofilaria immitis, recognized BmAl-1 on enzyme-linked immunosorbent assay and Western blots. BmAl-1 showed high binding affinity for a fatty acid; however, a search for sequence homology with known fatty acid binding proteins indicated that BmAl-1 is a unique fatty acid binding protein. This 35-kDa protein seems to be highly conserved in different stages and species of filarids, and it represents a previously unknown allergen that is possibly involved in the pathogenesis of filarial disease.     

Dipetalonema viteae and Brugia pahangi transplant infections in gerbils for use in antifilarial screening

Transplanted infections of Dipetalonema viteae and Brugia pahangi have been evaluated as tools for experimental chemotherapy. Attempts were made to establish these filariae in similar pharmacokinetic sites within the same host, so that direct comparisons of in vivo drug susceptibilities could be made. Unfortunately, it was not possible to establish B. pahangi in the subcutaneous tissues, the preferred site of D. viteae. Therefore, intraperitoneal B. pahangi and subcutaneously implanted D. viteae in gerbils were used for the study. D. viteae infections were significantly enhanced by concomitant infections with B. pahangi, while B. pahangi infection rates were unaffected by the presence of D. viteae. Experiments with amoscanate, CGP6140 and Mel W demonstrated the importance of employing both B. pahangi and D. viteae for antifilarial discovery work and the fundamental effect of parasite location on drug efficacy. D. viteae rapidly migrate from the peritoneal cavity of gerbils following implantation; twenty one hours after infection 73% of transplanted worms were found in the subcutaneous tissues. It was shown that the migration response could be used as a stringent parameter for demonstrating antifilarial activity. D. viteae were exposed to antifilarial drugs for 24 hours in vitro, washed and implanted into the peritoneal cavity of gerbils. At autopsy, 5 days later, 10(-8)M ivermectin and milbemycin D had prevented migration; CGP6140, amoscanate, suramin, flubendazole and furapyrimidone were also detected at less than 10(-6)M using this parameter. In all cases the migration response was more sensitive to drugs than parasite kill. Ivermectin's ability to inhibit worm migration through the tissues is discussed, with respect to the role of itinerant males in the reproductive cycle of Onchocerca volvulus.     

Studies with Brugia pahangi 10. An attempt to demonstrate the sharing of antigenic determinants between the worm and its hosts.

Infective stage Brugia pahangi that were reared in Aedes aegypti survived equally well in cats that had previously been immunized against mosquito tissue and in a normal cat. The survival of third, fourth, juvenile, adult and microfilarial stages of B. pahangi that were recovered from cats was similar in jirds that had been immunized against cat antigens and in normal jirds. Host antigenic determinants were not detected on the surface of larvae in substantial amounts using fluorescent antibody techniques. It is unlikely that B. pahangi evades the immune response of its vertebrate hosts by masquerading as "self" behind host antigens.     

Profiling the cellular immune response to multiple Brugia pahangi infections in a susceptible host

Human lymphatic filariasis is caused primarily by Brugia malayi and Wuchereria bancroffi. Unraveling this disease is complex, as people living in endemic areas exhibit a vast array of clinical states and immune responses. The Mongolian gerbil (Meriones unguiculatus)-B. pahangi model of human lymphatic filariasis has provided much information on immune parameters associated with filarial infection. Prior investigations in our laboratory have shown that gerbils closely mimic a subset of patients classified as microfilaremic but asymptomatic, a group that comprises the majority of people living in endemic areas. Worm recovery data suggest that gerbils carrying current B. pahangi infections do not show any resistance to subsequent subcutaneous B. pahangi infections. The aim of the present studies was to investigate the T cell cytokine response in gerbils receiving multiple infections of B. pahangi as a means of mimicking the conditions experienced by people in endemic areas. The T cell cytokine profile generated by multiply infected gerbils was not different from that previously generated by gerbils infected only once with B. pahangi. Gerbils infected multiple times with B. pahangi showed a transient increase in IL-5, which corresponded to the increased eosinophil levels previously reported from multiply infected gerbils. Chronically infected gerbils showed elevated IL-4 mRNA levels, as has been reported from gerbils infected only once with B. pahangi. Chronic infections were also associated with a state of immune hyporesponsiveness, as determined by the characterization of lymphatic thrombi and lymphoproliferation of spleen and renal lymph node cells to worm antigen.